Studies in Mali on Drug Resistance to Malaria To address the issue of antimalarial drug resistance in Mali, we completed a WHO-recommended, therapeutic efficacy study to estimate the effectiveness of artemether-lumefantrine (AL), the frontline treatment for uncomplicated Plasmodium falciparum malaria in Mali. We enrolled 146 unique children from Kenieroba, Mali, aged 2-17 years, presenting with uncomplicated falciparum malaria. These 146 children presented a total of 185 malaria episodes (36 and 3 children had a second and third malaria episode), We treated all subjects with directly-observed AL twice daily for 3 days. AL was safe and well tolerated; adverse events were mostly mild and mostly deemed unrelated, unlikely related, or possibly related to treatment. Three cases of vomiting occurred during dosing and 2 cases of severe malaria developed on protocol, which were reported as serious adverse events. During a 28-day follow-up period, we found 34 recurrent infections that were genotyped and determined to be new infections and not recrudescences. We found that AL is 100% effective and found no evidence for emerging ex vivo resistance to lumefantrine. Malaria Transmission Transmission of malaria is a critical aspect of the parasite life cycle but is poorly understood, and we have increased our efforts to investigate parasite sexual stages, malaria transmission in the field, and the biology of the mosquito vector. In May of 2014 we completed a clinical protocol studying 500 individuals of all ages in Kenieroba (13-I-N107). Volunteers were finger-pricked twice per month for a year to analyze both DNA and RNA. We first completed analysis of parasite DNA by PCR directly on filter paper, examining more than 10,000 samples. Using sensitive molecular methods rather than slide reading, we have found that a relatively high proportion of individuals of all ages carry malaria parasites prevalence was highest in November of 2013 during the wet season and lowest in May of 2014. Unlike most studies, we assessed longitudinal prevalence of parasites in the population and by a linear regression model, only age and gender showed significant effects on the longitudinal prevalence (p<0.0001 and p=0.0008, respectively), while other host factors did not. We showed for the first time that increasing P. falciparum longitudinal prevalence throughout the year was associated with decreasing risk of clinical malaria. This suggests that those with persistent parasite carriage acquire stronger protective immunity against clinical malaria. Using the parasite DNA barcoding technique and have found that most individuals in Kenieroba carry more than one clone of P. falciparum and that this distribution does not change from the wet season to the dry season. Further, using RT-PCR to test for gametocyte mRNA encoding Pfs25, we have determined that 50-80% of people that harbor parasite DNA also have detectable gametocytes throughout the year, showing that no one group can be uniquely targeted for interventions to reduce transmission. Screening of Malian Cohort for Antibodies to Asexual Stage Antigens We have also used the samples derived from the Malian cohort to study the responses of individuals to one of the major blood stage vaccine candidates PfRh5 and other proteins in its complex. This complex is critical for invasion of host red blood cells and promising clinical trial results have recently become available (S. Draper et al.). We have quantitated antibodies to PfRH5 and other complex proteins in Malians of various ages and established that the concentrations of anti-PfRh5 antibodies are far lower (100x less) than has been seen in a vaccine study in nave volunteers from the UK. The basis for this is not clear given the extensive exposure of the Malians but this is important information as a prelude to clinical trials in the field with members of this complex. Dry Season Ecology of Malaria Vectors Our main work continues to focus on: (1) Windborne long-range migration of mosquitoes as strategies of vector and malaria persistence in the Sahel, and (2) Mosquito dormancy (aestivation) through the dry season to account for the persistence of A. coluzzii . Building on previous results, we have amassed additional compelling evidence that A. coluzzii (previously, the M form of A. gambiae) persists locally by aestivation whereas A. gambiae (previously, the S form) and A. arabiensis re-colonize the area after the onset of rains. To explain the long-distance migration our project combines (i) on-the-ground monitoring of vector density and composition along 9 surveillance villages, spanning over 400 km, with (ii) aerial sampling (40-250 meters above ground) of mosquitoes using traps tethered to helium filled balloons. We continue to work on identification of the mosquitoes and many other insects that are caught in these high-altitude traps; to date this analysis has established the first report of wind-assisted long-distance migration of malaria vectors. These findings have important implications for malaria control and for the understanding and modeling of malaria transmission. Naturally Occurring Wolbachia in Anopheline Mosquitoes from Mali Wolbachia is a bacterium commonly found in arthropods that is known to reduce susceptibility of culicine mosquitoes to several Arboviral infections. Anopheline mosquitoes were thought to be naturally refractory to Wolbachia infection. However, we identified a naturally-occurring strain of Wolbachia in An. gambiae s.l. mosquitoes in the Malian villages of Kenieroba and Dangassa, different from strains previously identified in Burkina Faso. Field and laboratory data indicate that Wolbachia infection reduces the prevalence and intensity of sporozoite infection. We carried out experimental P. falciparum infections and found that Wolbachia does not affect the early stages of Plasmodium in the mosquito, but the prevalence and intensity of sporozoite infection was significantly reduced in females infected with Wolbachia (see PNAD publication). More recently we have trained a Malian investigator to do genomic DNA extractions from field-caught desiccated mosquitoes, and we have optimized a new single-PCR detection assay to detect Wolbachia. We will examine the seasonal variation in prevalence of infection in Kenieroba and Dangassa and survey other villages in the area to establish the range of distribution of Wolbachia in this region, as well as different larval habitats, to establish whether Wolbachia infection is widespread or limited to specific larval breeding sites. Leishmania in Mali Leishmania is another parasitic disease endemic to Mali. We have examined the impact of malaria control measures such as long-lived be nets and indoor residual spraying, showing that these interventions also impact sandflies and Leishmania infection. We have also done a study of the co-endemicity of filarial and Leishmania infections in different settings in Mali. The presence of the two infections transmitted by mosquitoes and sandflies makes integrated vector control very important in areas like these.